Hybridization assays may be employed for the detection and identification of DNA or RNA sequences. Published methods as used particularly in recombinant DNA research are described in Methods of Enzymology, Vol. 68, pp. 379-469 (1979); and Vol. 65, Part 1, pp. 468-478 (1968). One such method involving a preliminary separation of the nucleic acid fragments by electrophoresis is known as the "Southern Blot Filter Hybridization Method." See Southern E., J. Mol. Biol. 98, p. 503 (1975). A recent and more complete review of nucleic acid hybridization methods and procedures can be found in Meinkoth, J. and Wahl, G., Analytical Biochemistry, 138, pp. 267-284 (1984).
Fluorescent labeled synthetic polynucleotide probes are commercially available in the United States. Chemical methods for incorporating modified nucleotides into synthetic polynucleotides are described in the published PCT Application WO 84/03285, dated Aug. 30, 1985. The synthetic polynucleotide containing the modified nucleotide (usually referred to as a "linker arm nucleotide") can subsequently be derivatized with a fluorescent moiety. As described in the cited PCT application, only a single fluorescent moiety is attached to the probe.
Certain problems are encountered when using polynucleotide probes labelled with commonly available fluorophores such as fluorescein, rhodamine, pyrenes, etc. The most serious problem involves limited sensitivity for direct detection of the probe in the assay system. For most hybridization assays a sensitivity or detection level of at least 10.sup.-18 mole of labelled probe (10.sup.6 target molecules) is required. While many fluorophores inherently have this level of sensitivity, secondary interferences from the sample and components in the assay system prevent these levels of detection from being reached. At a level of 10.sup.-18 mole of fluorescent probe, fluorescence from the sample itself, Rayleigh scatter, reflection from support materials (nitrocellulose filters, etc.) and in particular Raman (water) scatter can produce background signals many orders of magnitude higher than the signal from the fluorescent probe.
Ideally, improvement in detecting fluorescent probes in such assay systems could be obtained by selecting a fluorophore which has: (1) a large Stokes shift, that is, a large separation between the wavelengths for maximum excitation (EX) and the wavelength for maximum emission (EM); (2) a high quantum yield (QY&gt;0.5); (3) a high extinction coefficient (EC&gt;30,000); (4) an emission beyond 600 nm (red fluorescence); and (5) an excitation maximum close to a laser line (442 nm Helium-Cadmium or 448 nm Argon). Unfortunately, there are no common fluorophores which fully satisfy these criteria. For example, fluorescein (EX: 495 nm, EM: 525 nm, QY=0.5) is a highly fluorescent label with an excitation maximum near a laser line, but has a Stokes shift of only .about.30 nm.
It is known that a larger Stokes shift can be obtained by employing a pair of donor/acceptor fluorophores which have overlapping spectra and which are arranged in close proximity for non-radiative energy transfer between the donor and acceptor fluorophores. This form of energy transfer was proposed by Forster, who developed equations of transfer efficiency in relation to separation distances between the fluorophores. See, for example, Forster, Th., Ann. Phys. (Leipzig) 2:55-75 (1948). A recent summary of Forster's non-radiative energy transfer is given in "Principles of Fluorescent Spectroscopy," J. R. Lakowicz, Chapt. 10 (1983). The Forster mathematical analysis predicates that the closer the spacing of the fluorescent moieties the greater the efficiency of energy transfer. Prior experimental evidence confirmed this prediction.
Stryer and Haugland (Proc. Natl. Acad. Sci. 58, 719-729, 1967) reported experiments with variable spacing for an energy donor and acceptor pair attached to oligopeptides. An energy donor group and an energy acceptor group were attached to the ends of proline oligomers which served as spacers of defined lengths. Spacings of 1 to 12 units were tested, with a separation range of 12 to 46 Angstroms (.ANG.). The longer oligomers were found to be in helical conformation. The energy transfer efficiency decreased from 100% at a distance of 12.ANG. to 16% at 46.ANG.. It was concluded that the dependence of the transfer efficiency on distance was in excellent agreement with the dependence predicted by the Forster equations. The results were so close to theoretical predictions that the authors proposed use of non-radiative energy transfer as a spectrocopic ruler. Related experiments with model systems reported by other researchers are confirmatory. See, for example, Gabor, Biopolymers 6:809-816 (1968); and Katchalski-Katzir, et al., Ann. N. Y. Acad. Sci. 366: 44-61 (1981). The use of the Forster energy transfer effect has been described in the following immunofluorescent assay patents. (See U. S. Pat. Nos. 3,996,345; 3,998,943; 4,160,016; 4,174,384; and 4,199,599). The energy transfer immunofluorescent assays described in these patents are based on the decrease or quenching of the donor fluorescence rather than fluorescent re-emission by the acceptor [Ullman, E. F., et al., J. Biol. Chem., Vol. 251, 14, pp. 4172-4178 (1976)].
Homogeneous immunoassay procedures based on chemiluminescent labels or bioluminescent proteins have been reported which involve non-radiative energy transfer, see Patel, et al., Clin. Chem. 29 (9):1604-1608 (1983); and European Patent Application No. 0 137 515, published Apr. 17, 1985. By close spacing of the donor-acceptor group according to the principles of nonradiative energy transfer for high transfer efficiency it was proposed that homogeneous assays could be made practical. Homogeneous assays are inherently simpler to carry out but their use had been subject to the limitation that unbound labelled probe remains in solution and causes interfering background signal. European Patent Application No. 0 137 515 published Apr. 17, 1985 refers to various ligand-ligand interactions which can be used with the bioluminescent proteins including nucleic acidnucleic acid interactions. The examples, however, are directed to protein ligands rather than nucleic acids.
European Patent Application No.,0 070 685, published Jan. 26, 1983, relates to homogeneous nucleic acid hybridization assays employing non-radioative energy transfer between absorber/emitter moieties positioned within 100 Angstroms of each other. As described, the hybridization probes are prepared by attaching the absorber-emitter moieties to the 3' and 5' end units of pairs of single-stranded polynucleotide fragments derived from DNA or RNA by restriction enzyme fragmentation. The pairs of polynucleotide fragments are selected to hybridize to adjacent complementary sequences of the target polynucleotide with the labelled ends with no overlap and with few or no base-pairing spaces left between them. The preferred donor moiety is a chemiluminescent catalyst and the absorber moiety is a fluorophore or phosphore.